RESUMO
Smokers (≥10 cigarettes per day, N=331) of European ancestry taking part in a double-blind placebo-controlled randomized trial of 12 weeks of treatment with bupropion along with counseling for smoking cessation were genotyped for a variable number of tandem repeats polymorphism in exon III of the dopamine D4 receptor gene. Generalized estimating equations predicting point-prevalence abstinence at end of treatment and 2, 6 and 12 months after the end of treatment indicated that bupropion (vs placebo) predicted increased odds of abstinence. The main effect of Genotype was not significant. A Genotype × Treatment interaction (P=0.005) showed that bupropion predicted increased odds of abstinence in long-allele carriers (odds ratios (OR)=1.31, P<0.0001), whereas bupropion was not associated with abstinence among short-allele homozygotes (OR=1.06, P=0.23). The Genotype × Treatment interaction remained when controlling for demographic and clinical covariates (P=0.01) and in analyses predicting continuous abstinence (P's≤0.054). Bupropion may be more efficacious for smokers who carry the long allele, which is relevant to personalized pharmacogenetic treatment approaches.
Assuntos
Bupropiona/uso terapêutico , Inibidores da Captação de Dopamina/uso terapêutico , Variação Genética , Receptores de Dopamina D4/genética , Abandono do Hábito de Fumar/métodos , Fumar/genética , Adulto , Bupropiona/farmacologia , Estudos Transversais , Inibidores da Captação de Dopamina/farmacologia , Método Duplo-Cego , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fumar/tratamento farmacológico , Resultado do TratamentoRESUMO
Certain junctions between ependymal cells, between astrocytes, and between some electrically coupled neurons have heretofore been regarded as tight, pentalaminar occlusions of the intercellular cleft. These junctions are now redefined in terms of their configuration after treatment of brain tissue in uranyl acetate before dehydration. Instead of a median dense lamina, they are bisected by a median gap 20-30 A wide which is continuous with the rest of the interspace. The patency of these "gap junctions" is further demonstrated by the penetration of horseradish peroxidase or lanthanum into the median gap, the latter tracer delineating there a polygonal substructure. However, either tracer can circumvent gap junctions because they are plaque-shaped rather than complete, circumferential belts. Tight junctions, which retain a pentalaminar appearance after uranyl acetate block treatment, are restricted primarily to the endothelium of parenchymal capillaries and the epithelium of the choroid plexus. They form rows of extensive, overlapping occlusions of the interspace and are neither circumvented nor penetrated by peroxidase and lanthanum. These junctions are morphologically distinguishable from the "labile" pentalaminar appositions which appear or disappear according to the preparative method and which do not interfere with the intercellular movement of tracers. Therefore, the interspaces of the brain are generally patent, allowing intercellular movement of colloidal materials. Endothelial and epithelial tight junctions occlude the interspaces between blood and parenchyma or cerebral ventricles, thereby constituting a structural basis for the blood-brain and blood-cerebrospinal fluid barriers.
Assuntos
Encéfalo/citologia , Membrana Celular , Animais , Barreira Hematoencefálica , Galinhas , Plexo Corióideo/citologia , Cyprinidae , Epêndima/citologia , Lantânio , Camundongos , Microscopia Eletrônica , Neuroglia/citologia , Neurônios/citologia , PeroxidasesRESUMO
Superior cervical ganglia, whose vessels are fenestrated and permeable to protein tracers such as horseradish peroxidase, were transplanted to undamaged surfaces in the fourth ventricle of rat pup brains. Horseradish peroxidase, infused systemically into the host, was exuded from the graft's vessels into the graft's extracellular stroma within 1 minute. At later times the glycoprotein reached the extracellular clefts of adjacent brain tissue, the vessels of which appeared to retain their impermeability. The blood-brain barrier to horseradish peroxide was thus bypassed where the extracellular compartments of graft and brain became confluent. The graft of autonomic ganglia can serve as a portal through which peptides, hormones, and immunoglobulins may likewise enter the brain.
Assuntos
Barreira Hematoencefálica , Ventrículos Cerebrais/fisiologia , Gânglios Autônomos/transplante , Animais , Gânglios Autônomos/irrigação sanguínea , Gânglios Autônomos/fisiologia , Peroxidase do Rábano Silvestre , RatosRESUMO
There are three distinct enolase isoenzymes in brain; neuron-specific enolase (NSE), formerly referred to as neuron-specific protein, which is specifically localized in neurons, a nonneuronal enolase (NNE), and a third hybrid form. Light microscopy with immunocytochemical techniques has permitted localization of non-neuronal enolase. The NNE is located in glial cells with no staining of endothelial cells or neurons. Thus, NSE and NNE can be used as specific metabolic markers for neurons and glial cells, respectively.
Assuntos
Encéfalo/enzimologia , Isoenzimas/metabolismo , Neuroglia/enzimologia , Neurônios/enzimologia , Fosfopiruvato Hidratase/metabolismo , Animais , Reações Cruzadas , Técnicas Imunoenzimáticas , Fígado/enzimologia , Fosfopiruvato Hidratase/imunologia , RatosRESUMO
The aspects presented here of how solutes, viruses and cells are able to cross the BBB indicate that there must be an active interaction of endothelium with viruses and immune system cells before they can penetrate the brain and spinal cord. The axoplasmic pathway taken by lectin-solute conjugates is similar but not identical to that followed by viral particles during their retrograde or anterograde transit through the axoplasm. Both the conjugates and virus are transferred to other neurons transsynaptically but the receptor mediated transfer utilized by viruses is far more specific. Cranial nerves are involved in both the entry and egress of antigens into and out of the brain. Antigen, generated within the CNS, may be able to escape from the brain to lymphoid tissue by passing into the fluid around a cranial nerve, thence via the lymph into lymph nodes to initiate an immune response involving the CNS.
Assuntos
Barreira Hematoencefálica , Fenômenos Fisiológicos Virais , Animais , Permeabilidade da Membrana Celular , Movimento Celular , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Encefalite/fisiopatologia , Encefalite/virologia , HIV-1/fisiologia , Humanos , Leucócitos/fisiologia , Leucócitos/virologia , Macrófagos/fisiologia , Macrófagos/virologia , Camundongos , Neurônios/virologia , Farmacocinética , Viremia/complicações , Viremia/fisiopatologiaRESUMO
Autonomic (superior cervical) ganglia, the vessels of which are freely permeable to macromolecules, from mature rat donors (allografts or autografts) were transplanted to different sites in the central nervous system (CNS). Minimal trauma was caused by grafts into the IVth ventricle while grafts to intraparenchymal locations such as cerebral cortex and spinal cord were necessarily traumatic and produced glial scarring. Postoperative periods were between 4 weeks and 30 months. A potentially significant aspect of neural transplantation is the functional vascular connections between host and graft. It is highly likely that grafting procedures alter the blood-brain barrier (BBB) in the recipient brain. In order to determine permanent BBB changes, the glycoprotein horseradish peroxidase (HRP) (M.W. 40,000) was injected intravascularly for circulation periods ranging between 50 seconds and 90 minutes. Protein exudation was monitored by using the chromogens DAB and the highly sensitive TMB. All autonomic ganglia transplants, regardless of postoperative or HRP circulation times, were permeable to the injected protein; no qualitative differences were found between allografts and autografts. The blood-borne protein traversed the autonomic graft and infiltrated into the host brain for distances between 200 micron in intraparenchymal grafts to over 1 mm in intraventricular grafts; a smaller exudate was found in the intraparenchymal model than in the intraventricular site probably due to glial scarring that impeded the protein movement in the interstitial spaces. Significantly, TMB demonstrated that the systemic protein entered the cerebrospinal fluid. HRP was detected on the ventricular floor and in the perivascular spaces of the microvasculature. Transplantation of an autonomic ganglion into the brain provides a biological portal that bypasses normal barriers to macromolecules. The vascular and extracellular confluences between host and graft could provide direct access for systematically administered substances to enter brain regions where they, normally, would be excluded.
Assuntos
Barreira Hematoencefálica , Gânglios Simpáticos/transplante , Animais , Ratos , Ratos Endogâmicos , Transplante Autólogo , Transplante HomólogoRESUMO
The multilamellate glial sheath of mixed nerve roots of the sixth abdominal ganglion of crayfish contains numerous hemi-desmosomes which appear to attach glial lamellae to material in adjacent extracellular clefts. These junctions, which have been described in detail in an earlier report (Shivers and Brightman, '76), are irregular in shape, punctuate and may be as large as 1 mum in diameter. Surgical interruption of sixth ganglion nerve roots results in regeneration of motor axons and their multilamellate glial sheaths. As the glial processes grow and re-establish a highly organized axon sheath, hemi-desmosomes appear. These junctions are present at the advancing edge of glial processes as well as on their lateral margins. Developing hemi-desmosomes are characterized as a diffuse aggregation of 120-130 A intramembrane particles which are present three weeks following nerve section. As growth and reorganization of the sheath proceeds, the intramembrane particles appear to aggregate and form irregular clusters of varying dimensions. Regenerating nerves freeze-cleaved 8 to 16 weeks following surgery exhibit junctional particle aggregates similar to those in normal unoperated nerve roots. Origin of the intramembrane particles which comprise the junctional aggregated in unknown. Perhaps they are synthesized de novo by the regenerating glial cells or, they may be remnants of complexes which became dispersed following surgery. This is the first report of a freeze-fracture study of hemi-desmosome plasticity in an invertebrate nervous system.
Assuntos
Desmossomos/fisiologia , Regeneração Nervosa , Neuroglia/ultraestrutura , Nervos Periféricos/fisiologia , Animais , Astacoidea , Desmossomos/ultraestrutura , Técnica de Fratura por Congelamento , Neuroglia/fisiologia , Nervos Periféricos/ultraestrutura , Fatores de TempoRESUMO
Autonomic preganglionic, sensory, and lower motoneuron perikarya within the central nervous system, as well as cell bodies with axons projecting to the circumventricular organs, are retrogradely labeled with horseradish peroxidase (HRP) delivered to their axon terminals by cerebral and extracerebral blood. Subsequent to vascular injection of HRP into mice, blood-borne peroxidase passes across permeable vessels in muscle, ganglia, and in all circumventricular organs except for the subcommissural organ in which no leak could be discerned. Brain parenchyma adjacent to each of the permeable circumventricular organs quickly becomes inundated with the protein. By four to six hours post-injection, this extracellular HRP reaction product has disappeared, and by eight hours perikarya of specific hypothalamic nuclei contain HRP-positive granules indicative of the intra-axonal retrograde transport of the protein. Hypothalamic neurons so labeled are presumed to send axons to such circumventricular organs as the median eminence or neurohypophysis and include neurons of the magnocellular neurosecretory supraoptic and paraventricular nuclei, the accessory magnocellular nuclei, the parvicellular arcuate nucleus, and a band of periventricular cells extending rostrally into the medial preoptic area. Labeled somata are also adjacent to the organum vasculosum of the lamina terminalis and in the vertical limb of the nucleus of the diagonal band of Broca. No similarly labeled cell bodies were identified near the subfornical organ.
Assuntos
Ventrículos Cerebrais/anatomia & histologia , Sistema Nervoso/enzimologia , Peroxidases/metabolismo , Animais , Transporte Axonal , Transporte Biológico , Barreira Hematoencefálica , Permeabilidade Capilar , Feminino , Histocitoquímica/métodos , Camundongos , Neurônios Aferentes , Peroxidases/sangueRESUMO
The sheath around the roots of the sixth abdominal ganglion in the ventral nerve cord of the crayfish consists of concentric layers of thin glial processes alternating with wide clefts containing filamentous connective tissue. Regions of each glial lamella are perforated by single, short, tubular channels: the trans-glial channels. In thin plastic sections examined in the electron microscope, the channels appear as slits that are 240 A wide and 450-550 A long which traverse glial lamellae less than 1,500 A thick. Branched tubular channels cross glial sheets that are thicker than 1,500 A. The thickest glial wrap is adaxonal; it closely encapsulates individual axons and its cell membrane is separated from the axolemma by a collagen-free space of only 150 A. The adaxonal glial cytoplasm contains unique, three-dimensional networks of interconnected tubules. Separate tubular lattices occur along these thicker processes. In replicas of freeze-fractured sheaths, the outer half of the plasma membrane belonging to the thin glial sheets exhibits many volcano-like protrusions which represent cross fractures through the necks of trans-glial channels. Corresponding depressions on the inner half of these membranes are sites where the plasma membrane invaginates to form the channels. Although some channels are randomly dispersed, others are lineraly positioned in restricted areas across successive glial layers. The number of channels is far more readily appreciated in replicas than in thin sections. The average frequency of channels is 16 per mu2 (range 8 to 33) in normal roots and does not differ significantly from the average of 13 per mu2 in proximal stumps of roots fixed three to four weeks after the roots were cut. The channels are not precisely aligned from one glial layer to the next but do appear to coincide approximately with the adaxonal tubular lattice. The combination of trans-glial channels and adaxonal tubular lattices may provide a complex conduit that could facilitate a rapid, passive flow of electrolytes and nutrients across the nerve sheath to the axonal surface. Horseradish peroxidase solutions bathing the ventral roots enter the trans-glial channels, extracellular clefts and finally the tubular lattices. This distribution supports the proposed role of the channels in a rapid extracellular passage of solutes. The channel profiles have a range of forms consistent with the supposition that they are not static but continually reforming. There are indications that, proximal to the cut, the areas of glial plasma membrane with channel profiles contain more junctional complexes between regenerating cells than between glial cells of normal sheaths. The channel profiles and aggregates of particles belonging to junctions are closely associated when they occupy the same region of the membrane.
Assuntos
Astacoidea/anatomia & histologia , Gânglios/ultraestrutura , Neuroglia/ultraestrutura , Animais , Junções Intercelulares/ultraestrutura , Regeneração NervosaRESUMO
Neurosecretory neurons of the hyperosmotically stressed hypothalamo-neurohypophysial system have been a useful model with which to demonstrate interrelationships among perikaryal lysosomes, agranular reticulum-like cisterns, endocytotic vacuoles, and the axoplasmic transport of acid hydrolases and horseradish peroxidase. Supraoptic neurons from normal mice and mice given 2% salt water to drink for 5--8 days have been studied using enzyme cytochemical techniques for peroxidase and lysosomal acid hydrolases. Peroxidase-labeling of these neurons was accomplished by intravenous injection or cerebral ventriculocisternal perfusion of the protein as previously reported (Broadwell and Brightman, '79). Compared to normal controls, supraoptic cell bodies from hyperosmotically stimulated mice contained elevated concentrations of peroxidase-labeled dense bodies demonstrated to be secondary lysosomes and acid hydrolase-positive and peroxidase-positive cisterns either attached or unattached to secondary lysosomes. These cisterns were smooth-surfaced and 400--1,000 A wide. Their morphology was similar to that of the agranular reticulum. Some of the cisterns contained both peroxidase and acid hydrolase activities. The cisterns probably represent an elongated form of lysosome and, therefore, are not elements of the agranular reticulum per se. By virtue of their direct connections with perikaryal secondary lysosomes, these cisterns may provide the route by which acid hydrolases and exogenous macromolecules can leave perikaryal secondary lysosomes for anterograde flow down the axon. Very few smooth-surfaced cisterns were involved in the retrograde transport of peroxidase within pituitary stalk axons from normal and salt-treated mice injected intravenously with peroxidase. Peroxidase undergoing retrograde transport was predominantly in endocytotic structures such as vacuoles and cup-shaped organelles, which deliver this exogenous macromolecule directly to secondary lysosomes for degradation in the cell body. These observations extend our previously reported findings in the axon to the cell body and suggest that agranular reticulum-like cisterns in the perikaryon, like those in the axon, may be part of the lysosomal system rather than associated with the agranular reticulum. A diagram summarizing the lysosomal system of organelles and proposed transport of acid hydrolases and peroxidase in neurosecretory neurons specifically and in neurons in general is provided.
Assuntos
Peroxidase do Rábano Silvestre/metabolismo , Hidrolases/metabolismo , Hipotálamo/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Peroxidases/metabolismo , Núcleo Supraóptico/metabolismo , Animais , Transporte Axonal , Transporte Biológico Ativo , Grânulos Citoplasmáticos/ultraestrutura , Feminino , Camundongos , Núcleo Supraóptico/ultraestruturaRESUMO
The meninges of various mammals were prepared for examination with the electronmicroscope by thin sectioning or freeze-fracturing. Particular attention was given to the distribution of tight junctions in order to determine the basis for the meningeal barrier between the blood circulating in dural vessels and the cerebrospinal fluid in the subarachnoid space. While some dural blood vessels are fenestrated, those in the subarachnoid space are not and their component endothelial cells are joined by an extensive system of tight junctions. An extensive and continuous system of tight junctions was also found in a layer of specialized cells at the border of the arachnoid with the dura. This arachnoid barrier layer is apparently the only basis of the meningeal barrier because often cellular layers in the dura and arachnoid lack tight junctions although they are linked by gap junctions and desmosomes. In particular, tight junctions are lacking at the border of the "subdural space" which is actually a fascial plane within the dura. Tight junctions are also lacking between astrocytes at the surface of the brain but these cells are linked by gap junctions and a new type of intercellular junction. The distribution of these junctions, as well as assemblies of intramembranous particles at the astrocytic border, raises the question whether this layer might have a role in the exchange of certain substances between the brain and cerebrospinal fluid.
Assuntos
Astrócitos/ultraestrutura , Junções Intercelulares/ultraestrutura , Meninges/ultraestrutura , Neuroglia/ultraestrutura , Animais , Aracnoide-Máter/ultraestrutura , Gatos , Chinchila , Desmossomos/ultraestrutura , Dura-Máter/ultraestrutura , Técnica de Fratura por Congelamento , Haplorrinos , Macaca , Camundongos , Microscopia Eletrônica , Pia-Máter/ultraestrutura , Coelhos , Ratos , Especificidade da EspécieRESUMO
The versatility of horseradish peroxidase is its usefulness both as an antigenic marker and as a probe molecule. We have demonstrated in the neuroendocrine cell that an HRP-bound antibody offers a high order of resolution for determining in which cellular compartment an antigen is located and where it is not. When native peroxidase is applied as an intracellular probe, it labels organelles associated with endocytosis in retrograde axonal transport and with the lysosomal system in both retrograde and orthograde axonal transport. The investigation that remains is the application of lectin-bound HRP to determine the pathways of membrane flow at the time when the neuroendocrine cell is stimulated to synthesize, transport, and secrete its peptide. For example, we are interested to know (1) whether internalized axon terminal membrane tagged with wheat germ agglutinin-HRP is channeled to all Golgi saccules engaged in the production of secretory granules in salt stimulated supraoptic neurons; and (2) if internalized cell membrane of the supraoptic cell body is tagged with wheat germ agglutinin-HRP and channeled to GERL, will this membrane be transferred from GERL to secretory granules, lysosomes in the cell body and axon, the axonal endoplasmic reticulum, and to autophagic/crinophagic vacuoles in axon terminals of salt-stressed supraoptic neurons? These additional studies should provide a more comprehensive, morphological picture of membrane flow in a neuroendocrine cell that is responding to the metabolic demands placed upon it.
Assuntos
Peroxidase do Rábano Silvestre , Neurossecreção , Sistemas Neurossecretores/metabolismo , Peroxidases , Animais , Transporte Axonal , Endocitose , Histocitoquímica , Peroxidase do Rábano Silvestre/metabolismo , Técnicas Imunoenzimáticas , Microscopia Eletrônica/métodos , Neurônios/fisiologia , Neurofisinas/análise , Sistemas Neurossecretores/ultraestruturaRESUMO
Treatment of cultured hippocampal neurons with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the absence of glucose mimics ischemic energy depletion and induces formation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) clusters, spherical structures with diameters of 75-175 nm [Dosemeci et al., J. Neurosci. 20 (2000) 3076-3084]. The demonstration that CaMKII clustering occurs in the intact, adult rat brain upon interruption of blood flow indicates that clustering is not confined to cell cultures. Application of N-methyl-D-aspartate (250 microM, 15 min) to hippocampal cultures also induces cluster formation, suggesting a role for Ca(2+). Indeed, intracellular Ca(2+) monitored with Fluo3-AM by confocal microscopy reaches a sustained high level within 5 min of CCCP treatment. The appearance of immunolabeled CaMKII clusters, detected by electron microscopy, follows the onset of the sustained increase in intracellular Ca(2+). Moreover, CaMKII does not cluster when the rise in intracellular Ca(2+) is prevented by the omission of extracellular Ca(2+) during CCCP treatment, confirming that clustering is Ca(2+)-dependent. A lag period of 1-2 min between the onset of high intracellular Ca(2+) levels and the formation of CaMKII clusters suggests that a sustained increase in Ca(2+) level is necessary for the clustering. CaMKII clusters disappear within 2 h of returning the cultures to normal incubation conditions, at which time no significant cell death is detected. These results indicate that pathological conditions that promote sustained episodes of Ca(2+) overload result in a transitory clustering of CaMKII into spherical structures. CaMKII clustering may represent a cellular defense mechanism to sequester a portion of the CaMKII pool, thereby preventing excessive protein phosphorylation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Metabolismo Energético/fisiologia , Hipocampo/enzimologia , Líquido Intracelular/enzimologia , Neurônios/enzimologia , Fatores Etários , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Técnicas de Cultura de Células , Células Cultivadas/enzimologia , Células Cultivadas/patologia , Células Cultivadas/ultraestrutura , Quelantes/farmacologia , Citoplasma/enzimologia , Citoplasma/patologia , Citoplasma/ultraestrutura , Metabolismo Energético/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/enzimologia , Feto , Hipocampo/patologia , Hipocampo/fisiopatologia , Imuno-Histoquímica , Líquido Intracelular/efeitos dos fármacos , Microscopia Eletrônica , N-Metilaspartato/farmacologia , Neurônios/patologia , Neurônios/ultraestrutura , Neurotoxinas/farmacologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Desacopladores/farmacologiaRESUMO
The efficacy with which circulating horseradish peroxidase (HRP) spreads from transplants into the brain's interstitial spaces (IS), was assessed by 3 factors: graft type, site and age. Pieces of skeletal muscle, skin or entire superior cervical ganglion (SCG) were inserted into the IV ventricle (ventricular) or substance of the brain (parenchymal). The age of the grafts, i.e. the intervals after transplantation, were 1, 3, 6 and 12 months. Generally, HRP spread into the IS to about the same extent from ventricular muscle and skin autografts--1 mm, but less from parenchymal SCG allografts--0.5 mm. The spread from all grafts--ventricular and parenchymal--diminished with time. Exudation distance from muscle was the same as that from skin grafts for the first 6 months, but by 1 year, the penetration was significantly greater from muscle than from skin transplants. The flow of HRP was more extensive from parenchymal SCG grafts than from parenchymal muscle and skin grafts at 6 and 12 months. In some of the 6 and 12 month old parenchymal grafts of muscle and skin, no detectable HRP was extravasated. HRP consistently penetrated the brain more deeply from ventricular skin and muscle grafts than from parenchymal ones because more tissue mass survived in ventricular than in parenchymal autografts. Though care was taken not to damage the brain surface during ventricular insertion, there was a consistent, vigorous, collateral sprouting of, as yet unidentified, cranial nerves. These sprouts innervated muscle and skin autografts which, consequently, were able to survive for at least 1 year and contained vessels permeable to HRP. Allografts of muscle between inbred strains did not become innervated, survived for only 2 months and contained the central, barrier type of vessels, but not their intrinsic, permeable type. Thus, it is the muscle cell or its basal lamina within muscle grafts that determines the type of surviving vessel. In SCG allografts, even when all their ganglion cells had disappeared, leaving only connective tissue, Schwann cells and their basal lamina, the ganglion's capillaries survived and remained permeable to HRP. Therefore, the characteristics of the SCG vessels are determined by the Schwann cell-fibroblast milieu rather than the neuronal one.
Assuntos
Barreira Hematoencefálica , Gânglios Simpáticos/transplante , Músculos/transplante , Transplante de Pele , Animais , Sobrevivência de Enxerto , Peroxidase do Rábano Silvestre/metabolismo , Microscopia Eletrônica , Ratos , Ratos EndogâmicosRESUMO
The enolase (EC 4.2.1.11) isoenzymes, neuron-specific enolase (NSE, gamma gamma) and non-neuronal enolase (NNE, alpha alpha), are markers for neurons and glia, respectively, in adult mammalian brain. In developing fetal and early postnatal brain, levels of non-neuronal enolase (NNE) are high. Neuron-specific enolase (NSE) appears only after neurogenesis begins in a given region and only slowly attains adult levels. Immunocytochemistry in developing rat and rhesus monkey brain reveals that proliferative zones that give rise to neurons are NNE(+). Thus, nerve cells must undergo a switch from NNE to NSE. In addition, study of neurons in cerebellum and neocortex reveals that they are NNE(+) during migration and only become NSE(+) in their final location, presumably after making full synaptic connections. Such migrating cells may contain hybrid enolase (alpha gamma) and some (e.g. cerebellar stellate/basket cells) may not completely switch over to NSE even in the adult. Neuron-specific enolase is not only a specific molecular marker for mature nerve cells, but is closely correlated to the differentiated state.
Assuntos
Encéfalo/enzimologia , Diferenciação Celular , Isoenzimas/metabolismo , Neurônios/enzimologia , Fosfopiruvato Hidratase/metabolismo , Animais , Encéfalo/citologia , Movimento Celular , Cerebelo/enzimologia , Haplorrinos , Macaca mulatta , Neurônios/citologia , Radioimunoensaio , Ratos , Medula Espinal/enzimologia , Córtex Visual/enzimologiaRESUMO
Neuron-specific enolase (NSE) is an isoenzyme of the glycolytic enzyme enolase (EC 4.2.1.11) which also has a muscle and liver isoenzyme. Previous work has shown NSE to be specifically localized to neurons and neuroendocrine cells, but the application of NSE as a marker for cell cultures has not been investigated. Primary culture of central nervous system tissue derived from mice have been used to study optimal fixation procedures. The results show that NSE can serve as a useful alternative to non-specific histochemical strains or strictly morphologic criteria for identifying nerve cells.
Assuntos
Fosfopiruvato Hidratase/metabolismo , Medula Espinal/enzimologia , Animais , Técnicas de Cultura , Gânglios Espinais/enzimologia , Técnicas Imunoenzimáticas , Camundongos , Neurônios/enzimologia , Neurônios/ultraestrutura , Medula Espinal/anatomia & histologiaRESUMO
Three purported means by which large solutes may penetrate the blood-brain barrier are: permeabilized tight junctions; vesicular transport; or channel formation across cerebral blood vessels. The role of vesicular transport has been questioned, in part, because many cytoplasmic vesicles are induced by aldehyde fixation. Cryofixation reduces this artefact and was used to see structural changes in frog cerebral endothelium made permeable to plasma solutes after perivascular exposure to hyperosmotic (3 M) urea, or injury with a cold probe (-50 degrees C). Some control and experimental frogs were made hypothermic so as to inhibit endocytosis and autolytic changes. The brains of some untreated controls were immerse-fixed in aldehydes. Other controls and all other brains of normothermic or hypothermic animals were rapidly frozen, then substituted with acetone-fixative. The interendothelial tight junctions separate partially or completely, after hyperosmotic exposure, in one third of the junctions. Blood-borne ferritin and Evans blue pass through some of the patent junctions. Junctional opening is caused by cell shrinkage, because the perimeter/area ratio of individual endothelial cells in the hyperosmotic group is significantly greater than in the control, due to a decreased area. Large 0.08-0.32-micron-wide invaginations or pits of the endothelial cell membrane characterize both cryofixed and aldehyde-fixed vessels. The pits often appear as isolated vesicles in the cytoplasm, but serial sections reveal that many communicate with either the capillary lumen or subendothelial space. No series of pits opened onto both lumen and space to form a transendothelial channel. The number of vesicles in aldehyde-fixed specimens is about 4 times greater (P less than 0.01) and in the cold injured, cryofixed brain capillary, about two times greater (P less than 0.01), than in the cryofixed control. Hyperosmotic exposure does not increase the number of pits. The permeabilization of anuran cerebral endothelium by hyperosmotic treatment or cold injury is thus by means of an intercellular rather than a transcellular route.
Assuntos
Encéfalo/irrigação sanguínea , Permeabilidade Capilar/efeitos dos fármacos , Hipotermia/fisiopatologia , Ureia/farmacologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Circulação Cerebrovascular , Temperatura Baixa , Endotélio/efeitos dos fármacos , Endotélio/fisiologia , Endotélio/ultraestrutura , Congelamento , Hipotermia/patologia , Junções Intercelulares/fisiologia , Junções Intercelulares/ultraestrutura , Microscopia Eletrônica , Concentração Osmolar , Rana esculentaRESUMO
Plasma membranes of mature rat astrocytes separated by differential centrifugation have been reported to be intact, based on electron microscopic examination of thin plastic sections. However, the effects of the separation procedure on the internal structure of the plasma membranes are unknown. The degree of membrane integrity is of concern to us since our goal is the separation of astrocytic plasma membranes and characterization of the specific intramembranous particle groups called assemblies. We have taken advantage of the astrocyte membrane-marker, the assembly, in order to monitor, by freeze-fracture, the identity of the separated astrocytes and the integrity of their cell membrane. Since some processes of an astrocyte contain assemblies whereas other processes of the same cell do not, it was also necessary to determine if processes with assemblies were separated by this technique. Astrocytic cell membranes were also examined to determine if trypsinization or the mechanical disruption steps of the separation affected the intramembranous particles. Freeze-fracture of the plasma membranes revealed that the particles were rearranged resulting in patches of clumped intramembranous particles and areas of bare membrane. The assemblies were rearranged rather than lost from the membrane since they could be identified among the clumped particles. More astrocytic plasma membranes contained non-clumped, normally distributed particles in the trypsin treated fractions. The non-trypsinized fractions had more damaged astrocytes with aggregated intramembranous particles and much more cellular debris. We interpret the findings for the non-trypsinized astrocytes as due to greater mechanical stress placed on the cells during tissue disruption. Trypsin treatment lessens this stress, thereby, tending to preserve the normal distribution of intramembranous particles.
Assuntos
Astrócitos/análise , Encéfalo/citologia , Animais , Astrócitos/ultraestrutura , Membrana Celular/ultraestrutura , Técnicas Citológicas , Técnica de Fratura por Congelamento , Ratos , Ratos EndogâmicosRESUMO
The purpose of this study was to determine whether the blood vessels of transplanted neural tissue retain their functional characteristics. Quantitative autoradiography was used to measure local blood flow (F) with iodoantipyrine and the blood-to-tissue transfer constant (K) with alpha-aminoisobutyric acid in superior cervical ganglion (SCG) allografted to the surface of ventricle IV and into the cerebellum of the same rat. The F of the intraparenchymal grafts was slightly lower than that of the intraventricular grafts; F decreased between 1 and 4 weeks in SCG grafts at both sites. The permeability-surface area (PS) product of the microvessels and extraction fraction of AIB were calculated from these results and indicated restricted transvascular passage of the amino acid in both the in situ and grafted SCG. Surface area (S) and average length (L) of the microvessels were determined morphometrically and their permeability (P) was calculated from these data. Although K and PS decreased in the grafts compared to in situ SCG, a comparable decrease in S indicated that P was similar for the microvessels of both in situ and 1-week-old SCG transplants: 3.5-4.3 x 10(-6) cm/s. Between 1 and 4 weeks after transplantation, the P of the microvessels decreased to approximately 1.6-2.3 x 10(-6) cm/s without any change in S. Thus, the blood vessels of SCG grafts within or upon the brain initially retain the functional attributes of in situ SCG microvessels, but the average permeability of the graft microvessels decreases to approximately one half of the initial value by 4 weeks after transplantation.
Assuntos
Circulação Cerebrovascular , Gânglios Autônomos/transplante , Gânglios Simpáticos/transplante , Fosfatase Alcalina/metabolismo , Animais , Ventrículos Cerebrais/irrigação sanguínea , Gânglios Autônomos/irrigação sanguínea , Gânglios Simpáticos/irrigação sanguínea , Gânglios Simpáticos/enzimologia , Permeabilidade , Ratos , Ratos Endogâmicos , Fluxo Sanguíneo Regional , Fatores de Tempo , Transplante HomólogoRESUMO
The route taken by lanthanum (MW 139) across cerebral endothelium was delineated when the blood-brain barrier was opened by RMP-7, a novel bradykinin agonist. Balb C mice were infused through a jugular vein with LaCl3 with or without RMP-7 (5 micrograms/kg). Ten minutes later, the brains were fixed with aldehydes and processed for electron microscopy. The patency of the junctions between endothelial cells was estimated by counting the number of junctions penetrated by LaCl3. Tracer penetrated the junctions in about 25% of microvessels in vehicle infused, control mice and about 58% in the RMP-7 group, where more junctions per vessel were also penetrated. The LaCl3 then penetrated the basal lamina in about 20% of all microvessels in the RMP-7 group, versus 0.50% in the control group. From the basal lamina, the tracer entered perivascular spaces in about 13% of all microvessels in the RMP-7 group and about 0.07% in the controls. Very few endocytic pits or vesicles in the RMP-7 group were labeled, so LaCl3 did not cross endothelium by transcytosis. The increased number of tight junctions penetrated by tracer and its spread into periendothelial basal lamina and interstitial clefts indicated, therefore, a paracellular route of exudation in the RMP-7 treated animals.